Amino-spiro-quinazoline compounds

ABSTRACT

There are provided novel compounds of formula (I)                    
     wherein A represents an aromatic carbocyclic ring or a 5- or 6-membered heterocyclic aromatic ring containing 1 to 3 heteroatoms which may be the same or different and are selected from O, N and S; X represents —(CH 2 ) n — wherein n represents zero or 1; and R 1 , R 2  and R 3  are as defined in the specification, and pharmaceutically acceptable salts thereof, and enantiomers and tautomers thereof; together with processes for their preparation, compositions containing them and their use in therapy. The compounds are inhibitors of nitric oxide synthase and are thereby particularly useful in the treatment or prophylaxis of inflammatory disease and pain.

This is a divisional of application Ser. No. 09/125,174, filed Aug. 11,1998, now U.S. Pat. No. 6,083,952, which is a 371 of PCT/SE98/101206,filed Jun. 22, 1998 the entire content of which is hereby incorporatedby reference in this application.

FIELD OF THE INVENTION

The present invention relates to novel compounds which areaminospiropiperidine quinazoline derivatives. The invention alsoconcerns related aspects including processes for the preparation of thecompounds, compositions containing them and their use aspharmaceuticals. There are also provided chemical intermediates usefulfor the production of the compounds.

BACKGROUND OF THE INVENTION

Nitric oxide is produced in mammalian cells from L-arginine by theaction of specific nitric oxide synthases (NOSs). These enzymes fallinto two distinct classes—constitutive NOS (cNOS) and inducible NOS(iNOS). At the present time, two constitutive NOSs and one inducible NOShave been identified. Of the constitutive NOSs, an endothelial enzyme(ecNOS) is involved with smooth muscle relaxation and the regulation ofblood pressure and blood flow, whereas the neuronal enzyme (ncNOS)serves as a neurotransmitter and appears to be involved in theregulation of various biological functions such as cerebral ischaemia.Inducible NOS has been particularly implicated in the pathogenesis ofinflammatory diseases. Regulation of these enzymes should thereforeoffer considerable potential in the treatment of a wide variety ofdisease states (J. E. Macdonald, Ann. Rep. Med. Chem., 1996, 31,221-230).

WO 97/14686 discloses, amongst other compounds, aminospiropiperidinequinazoline derivatives of the following formula:

wherein R³ represents various substituents, for use as pharmaceuticals.The treatment or prophylaxis of inflammatory conditions is disclosed asa particular pharmaceutical use.

DISCLOSURE OF THE INVENTION

According to the present invention, there is provided a compound offormula (I)

wherein:

R¹ and R² independently represent hydrogen, C1 to 6 alkyl, C2 to 6alkenyl, C2 to 6 alkynyl, C1 to 6 alkoxy, C1 to 6 alkylthio, halogen,hydroxy, trifluoromethyl or amino;

R³ represents one or more substituents independently selected fromhydrogen, C1 to 6 alkyl, C2 to 6 alkenyl, C2 to 6 alkynyl, C1 to 6alkoxy, C1 to 6 alkylthio, halogen, hydroxy, trifluoromethyl, amino,cyano, nitro, trifluoromethoxy, methanesulphonyl, sulphamoyl, —NR⁴R⁵,—COOR⁶, —CONR⁷R⁸, benzyloxy, phenyl, or a 5-membered heterocyclicaromatic ring containing 1 to 3 heteroatoms which may be the same ordifferent and are selected from O, N and S, which phenyl or 5-memberedheterocyclic aromatic ring is optionally substituted, the optionalsubstituents being C1 to 6 alkyl, halogen, cyano, nitro, hydroxy, C1 to6 alkoxy, trifluoromethyl and trifluoromethoxy;

R⁴, R⁵ and R⁶ independently represent hydrogen or C1 to 6 alkyl;

R⁷ and R⁸ independently represent hydrogen, C1 to 6 alkyl or phenyl,which phenyl is optionally substituted by one or more groupsindependently selected from C1 to 6 alkyl, halogen, cyano, nitro,hydroxy, C1 to 6 alkoxy, trifluoromethyl and trifluoromethoxy;

X represents —(CH₂)_(n)—, wherein n represents zero or 1; and

A represents an aromatic carbocyclic ring or a 5- or 6-memberedheterocyclic aromatic ring containing 1 to 3 heteroatoms which may bethe same or different and are selected from O, N and S;

or a pharmaceutically acceptable salt, enantiomer, racemate or tautomerthereof.

The compounds of formula (I) and their pharmaceutically acceptablesalts, enantiomers, racemates and tautomers have the advantage that theyare potent inhibitors of the inducible isoform of the enzyme nitricoxide synthase (iNOS) present in macrophages.

The invention further provides a process for the preparation of suchcompounds or a pharmaceutically acceptable salt, enantiomer, racemate ortautomer thereof.

According to the invention there is also provided a compound of formula(I), or a pharmaceutically acceptable salt, enantiomer, racemate ortautomer thereof, for use as a medicament.

Another aspect of the invention provides the use of a compound offormula (I) or a pharmaceutically acceptable salt, enantiomer, racemateor tautomer thereof, in the manufacture of a medicament, for thetreatment or prophylaxis of diseases or conditions in which inhibitionof nitric oxide synthase activity is beneficial.

A more particular aspect of the invention provides the use of a compoundof formula (I) or a pharmaceutically acceptable salt, enantiomer,racemate or tautomer thereof, in the manufacture of a medicament, forthe treatment or prophylaxis of inflammatory disease.

According to the invention, there is also provided a method of treating,or reducing the risk of, diseases or conditions in which inhibition ofnitric oxide synthase activity is beneficial which comprisesadministering to a person suffering from or at risk of, said disease orcondition, a therapeutically effective amount of a compound of formula(I) or a pharmaceutically acceptable salt, enantiomer, racemate ortautomer thereof.

More particularly, there is also provided a method of treating, orreducing the risk of, inflammatory disease in a person suffering from orat risk of, said disease, wherein the method comprises administering tothe person a therapeutically effective amount of a compound of formula(I) or a pharmaceutically acceptable salt, enantiomer, racemate ortautomer thereof.

The compounds of the present invention may also be used advantageouslyin combination with a second pharmaceutically active substance,particularly in combination with a selective inhibitor of the inducibleisoform of cyclooxygenase (COX-2). Thus, in a further aspect of theinvention there is provided the use of a compound of formula (I) or apharmaceutically acceptable salt, enantiomer, racemate or tautomerthereof, in combination with a COX-2 inhibitor for the treatment ofinflammation, inflammatory disease and inflammatory related disorders.And there is also provided a method of treating, or reducing the riskof, inflammation, inflammatory disease and inflammatory relateddisorders in a person suffering from or at risk of, said disease orcondition, wherein the method comprises administering to the person atherapeutically effective amount of a compound of formula (I) or apharmaceutically acceptable salt, enantiomer, racemate or tautomerthereof in combination with a COX-2 inhibitor.

Preferably, A in formula (I) represents a benzo ring.

Preferably, R¹ in formula (I) represents hydrogen. Alternatively, it ispreferred that R¹ in formula (I) represents fluoro.

Preferably, R² in formula (I) represents fluoro.

In another preferred embodiment, n in formula (I) has a value of zero.

Particular compounds of the invention include:

(R*R*)-4′-amino-5′-fluoro-1,3,4,10a-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)quinazoline]-6-one;

(R*S*)-4′-amino-5′-fluoro-1,3,4,10a-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)quinazoline]-6-one;

(R*R*)-4-amino-5-fluoro-3b′,4′,6′,7′-tetrahydrospiro[quinazoline-2,5′(2H,9′H)-thieno[3,2-a]indolizine]-9′-one;

(R*S*)-4-amino-5-fluoro-3b′,4′,6′,7′-tetrahydrospiro[quinazoline-2,5′(2H,9′H)-thieno[3,2-a]indolizine]-9′-one;

(R*R*)-4-amino-5-fluoro-6′,7′,9′,9a-tetydrospiro[quinazoae-2,8′(2H,4′H)-thieno[2,3-a]indolizine]-4′-one;

(R*S*)-4-amino-5-fluoro-6′,7′,9′,9a′-tetrahydrospiro[quinoline-2,8′(2H,4′H-thieno[2,3-a]indolizine]-4′-one;

(R*R*)-4′-amino-7-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*S*)-4′-amino-7-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*R*)-4′-amino-8-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*S*)-4′-amino-8-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*R*)-4′-amino-9-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*S*)-4-amino-9-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*R*)-4′-amino-10-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*S*)-4′-amino-10-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*R*)-4′-amino-7,8-dichloro-5′-fluoro-1,3,4,10b-tetahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*S*)-4′-amino-7,8-dichloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*R*)-4′-amino-7-methyl-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*S*)-4′-amino-7-methyl-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*R*)-4′-amino-8-methyl-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*S*)-4′-amino-8-methyl-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*R*)-4′-amino-8-methoxy-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*S*)-4′-amino-8-methoxy-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*R*)-4′-amino-8-cyano-5′-fluoro-1,3,4,10b-tetahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*S*)-4′-amino-8-cyano-5′-fluoro-1,3,4,10b-tetahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*R*)-4′-amino-8-cyano-5′,8′-difluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*S*)-4′-amino-8-cyano-5′,8′-difluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one;

(R*R*)-4′-amino-5′-fluoro-7,8,10,10a-tetrahydrospiro[pyrido[3,4-a]indolizine-9,2′(1′H)-quinazoline]-5-one;

(R*S*)-4′-amino-5′-fluoro-7,8,10,10a-tetrahydrospiro[pyrido[3,4-a]indolizine-9,2′(1′H)-quinazoline]-5-one;

(R*R*)-4′-amino-5′-fluoro-6b,7,9,10-tetrahydrospiro[naphtho[2,1-a]indolizine-8,2′(1′H)-quinazoline]-12-one;

(R*S*)-4′-amino-5′-fluoro-6b,7,9,10-tetrahydrospiro[naphtho[2,1-a]indolizine-8,2′(1′H)-quinazoline]-12-one;

and pharmaceutically acceptable salts, enantiomers, racemates ortautomers thereof.

Unless otherwise indicated, the term “C1 to 6 alkyl” referred to hereindenotes a straight or branched chain alkyl group having from 1 to 6carbon atoms or a cyclic alkyl group having is from 3 to 6 carbon atoms.Examples of such groups include methyl, ethyl, n-propyl, i-propyl,n-butyl, i-butyl, t-butyl, cyclopentyl and cyclohexyl.

Unless otherwise indicated, the term “C2 to 6 alkenyl” referred toherein denotes a straight or branched chain alkyl group having from 2 to6 carbon atoms and including one double bond or a cyclic alkyl grouphaving from 3 to 6 carbon atoms and including one double bond. Examplesof such groups include ethenyl, 1- and 2-propenyl, 2-methyl-2-propenyl,2-butenyl, cyclopentenyl and cyclohexenyl.

Unless otherwise indicated, the term “C2 to 6 alkynyl” referred toherein denotes a straight or branched chain alkyl group having from 2 to6 carbon atoms and including one triple bond. Examples of such groupsinclude ethynyl, 1- and 2-propynyl and 2-butynyl.

Unless otherwise indicated, the term “C1 to 6 alkoxy” referred to hereindenotes a straight or branched chain alkoxy group having from 1 to 6carbon atoms. Examples of such groups include methoxy, ethoxy,n-propoxy, i-propoxy, n-butoxy, i-butoxy, s-butoxy and t-butoxy.

Other groups, for example, alkylthio, are to be interpreted similarly.

The process mentioned above, for the preparation of compounds of theinvention, or a pharmaceutically acceptable salt, enantiomer, racemateor tautomer thereof comprises reaction of a compound of formula (II)

wherein R¹ and R² are as defined above,

with a compound of formula (III) or a protected derivative thereof

wherein R³, A and X are as defined above.

The reaction may be carried out in a polar solvent, for example,methanol, ethanol, acetonitrile, dimethylformamide or dimethylsulphoxideat a suitable temperature, generally between 20° C. and the boilingpoint of the solvent, or without solvent at a temperature generallybetween 20° C. and 200° C. We have found that it is sometimes convenientto use the compounds of formula (III) in a protected form, for exampleas an acetal such as the diethoxy acetal. The process is then preferablycarried out in the presence of an acid catalyst. The required acetalsmay be formed by reacting an unprotected compound of formula (III) withan alcohol such as ethanol using methods that are well known in the art.

The present invention includes compounds of formula (I) in the form ofsalts, in particular acid addition salts. Suitable salts include thoseformed with both organic and inorganic acids. Such acid addition saltswill normally be pharmaceutically acceptable although salts ofnon-pharmaceutically acceptable acids may be of utility in thepreparation and purification of the compound in question. Thus,preferred salts include those formed from hydrochloric, hydrobromic,sulphuric, phosphoric, citric, tartaric, lactic, pyruvic, acetic,succinic, fumaric, maleic, methanesulphonic and benzenesulphonic acids.

Salts of compounds of formula (I) may be formed by reacting the freebase, or a salt, enantiomer, racemate or tautomer thereof, with one ormore equivalents of the appropriate acid. The reaction may be carriedout in a solvent or medium in which the salt is insoluble or in asolvent in which the salt is soluble, for example, water, dioxane,ethanol, tetrahydrofuran or diethyl ether, or a mixture of solvents,which may be removed in vacuo or by freeze drying. The reaction may alsobe a metathetical process or it may be carried out on an ion exchangeresin.

Novel intermediates of formulae (II) and (III) form another aspect ofthe invention.

Compounds of formula (II) may be prepared using methods such as thosedescribed in WO 97/14686.

The preparation of compounds of formula (III) is either known per se ormay be achieved using methods well known in the art. For example,compounds of formula (III) may be obtained by cyclisation of a compoundof formula (IV)

wherein R³, A and X are as defined above and M is an alkali or alkaliearth metal. Examples of such metals M include lithium, sodium,potassium or magnesium.

Compounds of formula (IV) may be formed in situ by treatment of acompound of formula (IV′)

wherein Hal represents a halogen atom, particularly bromine or iodine,with the metal M or an organometallic derivative or salt thereof, usingprocedures well known to the art. The reactions are best carried out inan aprotic solvent such as ether, tetrahydrofuran or diglyme attemperatures between −100° C. and 30° C.

Compounds of formula (IV′) may be prepared by reaction of the compoundof formula (V)

with an acid derivative of formula (VI)

wherein R³, A, X and Hal are as defined above, and L is a leaving group.The reaction may be performed in an organic solvent, for exampleethanol, dichloromethane or dimethylformamide at a temperature between0° C. and the boiling point of the solvent The reaction may be catalysedby the addition of a base; bases that may be used include organic amines(for example, triethylamine or pyridine) and alkali metal hydroxides,alkoxides or hydrides. Suitable leaving groups include halogen(especially chlorine) and imidazole. These acid derivatives may beprepared from the corresponding acid (compounds of formula (VI) whereinL is OH) using methods well known in the art.

Intermediate compounds may be used in protected form. Protecting groupsand details of processes for their removal may be found by reference tothe standard text “Protecting groups in Organic Synthesis”, 2nd Edition(1991) by Greene and Wuts.

The compounds of the invention and intermediates thereto may be isolatedfrom their reaction mixtures and, if necessary further purified, byusing standard techniques.

The compounds of formula I may exist in enantiomeric forms. Therefore,all enantiomers, diastereomers, racemates and mixtures thereof areincluded within the scope of the invention. The various optical isomersmay be isolated by separation of a racemic mixture of the compoundsusing conventional techniques, for example, fractional crystallisation,or HPLC.

Intermediate compounds may also exist in enantiomeric forms and may beused as purified enantiomers, diastereomers, racemates or mixtures.

The compounds of formula (I) may exist in alternative tautomeric forms.Compounds of formula (I) are provided in another tautomeric form or as amixture thereof.

The compounds of formula (I), and their pharmaceutically acceptablesalts, enantiomers, racemates and tautomers, are useful because theypossess pharmacological activity in animals. In particular, thecompounds are active as inhibitors of the enzyme nitric oxide syntase.More particularly, they are inhibitors of the inducible isoform of theenzyme nitric oxide synthase present in macrophages and as such arepredicted to be useful in therapy, for example, as anti-inflammatoryagents.

The compounds and their pharmaceutically acceptable salts, enantiomers,racemates and tautomers are indicated for use in the treatment orprophylaxis of diseases or conditions in which synthesis or oversynthesis of nitric oxide synthase forms a contributory part. Inparticular, the compounds are indicated for use in the treatment ofinflammatory conditions in mammals including man.

Conditions that may be specifically mentioned are:

osteoarthritis, rheumatoid arthritis, rheumatoid spondylitis, goutyarthritis and other arthritic conditions, inflamed joints;

eczema, psoriasis, dermatitis or other inflammatory skin conditions suchas sunburn;

inflammatory eye conditions including uveitis and conjunctivitis;

lung disorders in which inflammation is involved, for example, asthma,bronchitis, pigeon fancier's disease, farmer's lung, acute respiratorydistress syndrome;

bacteraemia, endotoxaemia (septic shock), aphthous ulcers, gingivitis,pyresis, pain and pancreatitis;

conditions of the gastrointestnal tract including Crohn's disease,atrophic gastritis, gastritis varialoforne, ulcerative colitis, coeliacdisease, regional ileitis, peptic ulceration, irritable bowel syndrome,damage to the gastrointestinal tract resulting from infections by, forexample, Helicobacter pylori, or from treatments with non-steroidalanti-inflammatory drugs;

and other conditions associated with inflammation.

The compounds will also be useful in the treatment and alleviation ofacute or persistent inflammatory or neuropathic pain or pain of acentral origin.

The compounds of formula (I) and their pharmaceutically acceptablesalts, enantiomers, racemates and tautomers may also be useful in thetreatment or prophylaxis of diseases or conditions in addition to thosementioned above. For example, the compounds may be useful in thetreatment of atherosclerosis, cystic fibrosis, hypotension associatedwith septic and/or toxic shock, in the treatment of dysfunction of theimmune system, as an adjuvant to short-term immunosuppression in organtransplant therapy, in the treatment of vascular complicationsassociated with diabetes and in cotherapy with cytokines, for exampleTNF or interleukins.

The compounds of formula (I) may also show inhibitory activity againstthe neuronal isoform of nitric oxide synthase. Thus they may also beuseful in the treatment of hypoxia, for example in cases of cardiacarrest and stroke, neurodegenerative disorders including nervedegeneration and/or nerve necrosis in disorders such as hypoxia,hypoglycemia, epilepsy, and in external wounds (such as spinal cord andhead injury), hyperbaric oxygen convulsions and toxicity, dementia, forexample pre-senile dementia, Alzheimer's disease and AIDS-relateddementia, Sydenham's chorea, Parkinson's disease, Tourette's Syndrome,Huntington's disease, Amyotrophic Lateral Sclerosis, Korsakoffs disease,imbecility relating to a cerebral vessel disorder, sleeping disorders,schizophrenia, depression, autism, seasonal affective disorder, jet-lag,depression or other symptoms associated with Premenual Syndrome (PMS),anxiety and septic shock Compounds of formula (I) may also be expectedto show activity in the prevention and reversal of tolerance to opiatesand diazepines, treatment of drug addiction, treatment of migraine andother vascular headaches, neurogenic inflammation, in the treatment ofgastrointestinal motility disorders, cancer and in the induction oflabour.

For the above mentioned therapeutic indications, the dosage administeredwill, of course, vary with the compound employed, the mode ofadministration and the treatment desired.

However, in general, satisfactory results are obtained when thecompounds are administered at a dosage of the solid form of between 1 mgand 2000 mg per day.

The compounds of formula (I), and pharmaceutically acceptablederivatives thereof, may be used on their own, or in the form ofappropriate pharmaceutical compositions in which the compound orderivative is in admixture with a pharmaceutically acceptable adjuvant,diluent or carrier. Administration may be by, but is not limited to,enteral (including oral, sublingual or rectal), intranasal, intravenous,topical or other parenteral routes. Conventional procedures for theselection and preparation of suitable pharmaceutical formulations aredescribed in, for example, “Pharmaceuticals—The Science of Dosage FormDesigns”, M. E. Aulton, Churchill Livingstone, 1988. The pharmaceuticalcomposition preferably comprises less than 80% and more preferably lessthan 50% of a compound of formula (I), or a pharmaceutically acceptablesalt, enantiomer, racemate or tautomer thereof.

There is also provided a process for the preparation of such apharmaceutical composition which comprises mixing the ingredients.

The compounds of formula (I), and pharmaceutically acceptablederivatives thereof, may also be advantageously used in combination witha COX-2 inhibitor. Particularly preferred COX-2 inhibitors are Celecoxiband MK-966. The NOS inhibitor and the COX-2 inhibitor may either beformulated together within the same pharmaceutical composition foradministration in a single dosage unit, or each component may beindividually formulated such that separate dosages may be administeredeither simultaneously or sequentially.

The invention is illustrated, but in no way limited, by the followingexamples:

PREPARATION 1

2,3-Dichloro-6-iodobenzoic Acid Chloride

a) 2,3-Dichloro-6-iodobenzoic acid

n-Butyllithium (1.45M solution in hexane, 19.4 mmol, 13.4 ml) was addeddropwise to a stirred solution of diisopropylamine (21.4 mmol, 3.0 ml)in THF (90 ml) at 0° C. After 30 minutes, the solution was cooled to−78° C. and a solution of 1,2-dichloro-4-iodobenzene (5.30 g, 19.4 mmol)in TIF (10 ml) was added dropwise. After 10 minutes, solid carbondioxide was added cautiously and the allowed to warm to room temperatureover 16 h. The mixture was diluted with water and extracted twice withethyl acetate. The aqueous layer was acidified with dilute aqueoushydrochloric acid and then extracted twice with ethyl acetate. Theextracts were dried over sodium sulphate and evaporated to give thetitle compound as a yellow solid (3.59 g). 300 MHz ¹H NMR (d₆-DMSO) 11.5(1H, br.s), 7.82 (1H, d, J 8.4 Hz), 7.44 (1H, d, J 8.4 Hz).

b) 2,3-Dichloro-6-iodobenzoic acid chloride

DMF (one drop) was added to a stirred solution of2,3-dichloro-6-iodobenzoic acid (1.53 g, 4.83 mmol) and oxalyl chloride(2 ml) in ethyl acetate (20 ml). After 2 hours, the solution wasevaporated and the product was used immediately.

PREPARATION 2

3-Cyano-6-iodobenzoic Acid Chloride

a) 2-Amino-5-cyanobenzoic acid methyl ester

2-Amino-5-bromobenzoic acid methyl ester (4.6 g, 20 mmol) and copper (I)cyanide (3.6 g, 40 mmol) were heated at 200° C. in N-methylpyrrolidinone(20 ml) for 4 h. After cooling to room temperature, the solution wasdiluted with water, extracted with ethyl acetate, the aqueous extractacidified with 2N hydrochloric acid and filtered. The organic layer wasseparated, dried over magnesium sulphate and evaporated. The residue waspurified by flash column chromatography on silica eluting with 50% ethylacetate in hexane to yield the title compound as a colourless oil (1.0g). MS (+El) ^(m)/z 176 [M⁺].

b) 2-Iodo-5-cyanobenzoic acid methyl ester

Sodium nitrite (0.8 g, 11.4 mmol) in water (10 ml) was added dropwiseover 10 minutes to a stirred solution of 2-amino-5-cyanobenzoic acidmethyl ester (2.0 g, 11.4 mmol) in water (30 ml) and conc. sulphuricacid (15 ml) at 0° C. After a fiber 15 minutes at 5° C., a solution ofpotassium iodide (3.5 g, 21 mmol) in water (30 ml) was quickly added andthe mixture allowed to warm to room temperature. 10% Aqueous sodiumthiosulphate was added, the mixture extracted with ethyl acetate, theextract dried over magnesium sulphate and evaporated. The residue waspurified by flash column chromatography on silica eluting with 20% ethylacetate in hexane to yield the title compound as a colourless oil (2.8g). MS (+EI) ^(m)/z 287 [M⁺].

c) 2-Iodo-5-cyanobenzoic acid

A solution of sodium hydroxide (0.44 g, 11 mmol) in water (10 ml) wasadded to 2-iodo-5-cyanobenzoic acid methyl ester (2.7 g, 9.4 mmol) inmethanol. The mixture was stirred for 20 h, then most of the solvent wasevaporated and the residue acidified with 2N hydrochloric acid. Theresulting precipitate was filtered off and dried to give a white solid(1.7 g). MS (−CI) ^(m)/z 272 [M-H]⁺.

d) 2-Iodo-5-cyanobenzoic acid chloride

This was prepared by the method of Preparation 1(b) and was useddirectly.

Other acid chlorides are either known per se or were prepared from theknown acids by the method of Preparation 1(b).

PREPARATION 3

1-(2-Bromo-6-chlorobenzoyl)-2,3-dihydro-4-pyridinone

A solution of 2-bromo-6-chlorobenzoic acid chloride (0.5 mmol) indichloromethane (5 ml) was added dropwise to a solution of2,3-dihydro-4-pyridinone (5.1 mmol, 495 mg) and triethylamine (1.2 ml)in dichloromethane (20 ml). After stirring for 30 minutes, the solutionwas diluted with water, separated, the aqueous extract further extractedwith dichloromethane, the combined organic extracts combined and washedwith brine, dried over sodium sulphate and evaporated. The residue waspurified by flash column chromatography on silica eluting with 30% ethylacetate in hexane to yield the title compound as a pale yellow oil (629mg). MS (+CI) ^(m)/z 316 [M+H]⁺.

The following intermediates were similarly prepared from2,3-dihydro-4-pyridinone and the appropriate acid chloride:

(i) 1-(2-Bromo-5-methylbenzoyl)-2,3-dihydro-4-pyridinone: colourless oilMS (+EI) ^(m)/z 293/295 [M⁺].

(ii) 1-(3-Bromo-4-pyridinoyl)-2,3-dihydro-4-pyridinone: white solid, MS(+EI) ^(m)/z 280/282 [M⁺].

(iii) 1-(2,3-Dichloro-6-iodobenzoyl)-2,3-dihydro-4-pyridinone: whitesolid, MS (+EI) ^(m)/z 395/397 [M⁺].

(iv) 1-(5-Cyano-2-iodobenzoyl)-2,3-dihydro-4-pyridinone: colourless oil,used without purification.

(v) 1-(2-Methyl-6-iodobenzoyl)-2,3-dihydro-4-pyridinone: MS (+EI) ^(m)/z341 [M⁺].

(vi) 1-(2-Bromo-3-chlorobenzoyl)-2,3-dihydro-4-pyridinone: MS (+EI)^(m)/z 313/315 [M⁺].

(vii) 1-(2-Bromo-4-chlorobenzoyl)-2,3-dihydro-4-pyridinone: MS (+EI)^(m)/z 314 [M⁺].

(viii) 1-(2-Bromo-5-chlorobenzoyl)-2,3-dihydro-4-pyridinone: MS (+EI)^(m)/z 314 [M⁺].

(ix) 1-(2-Bromo-3-thienoyl)-2,3-dihydro-4-pyridinone: MS (−CI) ^(m)/z284/286 [M−H]⁺.

(x) 1-(2-Bromo-5-methoxybenzoyl)-2,3-dihydro-4-pyridinone; MS (+EI)^(m)/z 309/311 [M⁺].

(xi) 1-(2-Iodo-1-naphthoyl)-2,3-dihydro-4-pyridinone: MS (−CI) ^(m)/z376 [M−H]⁺.

(xii) 1-(2-Iodo-3-thienoyl)-2,3-dihydro-4-pyridinone: colourless oil:300 MHz ¹H NMR (d₆-DMSO) 7.55 (1H, d, J 5.4 Hz), 7.47-7.43 (1H, m), 7.00(1H, d, J 5.4 Hz), 5.40 (1H, d, J 8.1 Hz), 4.18 (2H, t, J 7.2 Hz), 2.68(2H, t, J 7.2 Hz).

PREPARATION 4(±)-7-Chloro-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione

A mixture of 1-(2-bromo-chlorobenzoyl)-2,3-dihydro-4-pyridinone(Preparation 3) (300 mg, 0.954 mmol), triethylamine (0.39 mmol, 0.54ml), formic acid (0.05 ml, 1.3 mmol) and di(triphenylphosphine)diacetoxypalladium (47 mg, 0.063 mmol) in DMF (3 ml) was heated to 80°C. for 2 h. The cooled solution was filtered through celite, dilutedwith water, extracted twice with ethyl acetate, the extracts dried oversodium sulphate and evaporated. The residue was purified by flash columnchromatography on silica eluting with 50% ethyl acetate in hexane toyield the title compound as a colourless oil (70 mg). MS (+CI) ^(m)/z235/237 [M+H]⁺.

The compounds of Preparations 5 to 7 were synthesised using the methodof Preparation 4:

PREPARATION 5(±)-7,8-Dichloro-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione

From 1-(2,3-dichloro-6-iodobenzoyl)-2,3-dihydro-4-pyridinone. Whitesolid. MS (+El) ^(m)/z 269/271 [M⁺].

PREPARATION 6(±)-8-Chloro-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione

From 1-(2-bromo-5-chlorobenzoyl)-2,3-dihydro-4pyridinone. Colourlessoil. MS (+EI) ^(m)/z 235/237 [M⁺].

PREPARATION 7 (±)-3b,4,6,7-tetrahydrothieno[3,2-a]indolizine-5,9-dione

From 1-(3-bromo-2-thienoyl)-2,3-dihydro-4-pyridinone. Colourless oil. MS(+CI) ^(m)/z 208 [M+H]⁺.

PREPARATION 8(±)-8-Methyl-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione

A mixture of 1-(2-bromo-5-methylbenzoyl)-2,3-dihydro-4-pyridinone (1.19g, 4.03 mmol), tri-n-butyltin hydride (1.14 ml, 4.24 mmol) andazobis(isobutyronitrile) (AIBN, 66 mg, 10 mol %) in toluene (40 ml) washeated at 95° C. for 2 h, cooled, evaporated and purified by flashcolumn chromatography on silica eluting with 30% ethyl acetate in hexaneto yield the title compound as a pale yellow oil (276 mg), MS (+EI)^(m)/z 215 [M⁺].

The compounds of Preparations 9 to 12 were prepared using the method ofPreparation 8:

PREPARATION 9(±)-8-Cyano-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione:

From 1-(5-cyano-2-iodobenzoyl)-2,3-dihydro-4-pyridinone. MS (−CI) ^(m)/z226 [M⁺].

PREPARATION 10(±)-7,8,10,10a-Tetrahydropyrido[3,4-a]indolizine-5,9-dione

From 1-(3-bromo-4-pyridinoyl)-2,3-dihydro-4-pyridinone. MS (+EI) ^(m)/z202 [M⁺].

PREPARATION 11(±)-7-Methyl-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione

From 1-(2-Methyl-6-iodobenzoyl)-2,3-dihydro-4-pyridinone. MS (+EI)^(m)/z 215 [M⁺].

PREPARATION 12(±)-6b,7,9,10-tetraydronaphtho[2,1-a]indolizine-8,12-dione

From 1-(2-iodo-1-naphthoyl)-2,3-dihydro-4-pyridinone. MS (+CI) ^(m)/z252 [M+H]⁺.

PREPARATION 13 (±)-8-Methoxy-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione

Butyl lithium (3.1 ml, 0.9 M in hexanes, 2.8 mmol) was added dropwise toa solution of 1-(2-bromo-5-methoxybenzoyl)-2,3dihydro-4-pyridinone (0.75g, 2.4 mmol) in THF (20 ml) at −78° C. After stirring for 2 h, thereaction was quenched with water, extracted with ethyl acetate, theextract dried over magnesium sulphate and evaporated. The residue waspurified by flash column chromatography on silica eluting with 50% ethylacetate in hexane to yield the title compound as a colourless oil (240mg). MS (+EI) ^(m)/z 231 [M⁺].

The compounds of Preparations 14 to 16 were prepared using the method ofPreparation 13:

PREPARATION 1410-Chloro-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione

From 1-(2-bromo-3-chlorobenzoyl)-2,3-dihydro-4-pyridinone: colourlessoil. MS (+EI) ^(m)/z 235/237 [M⁺].

PREPARATION 159-Chloro-1,3,4,10b-tethydropyrido[2,1-a]isoindole-2,6-dione

From 1-(2-bromo-4-chlorobenzoyl-2,3-dihydro-4-pyridinone: colourlessoil. MS (+EI) ^(m)/z 235/237 [M⁺].

PREPARATION 16 6,7,9,9a-Tetrahydrothieno[2,3-a]indolizine-4,8-dione

From 1-(2-bromo-3-thienoyl)-2,3-dihydro-4-pyridinone. MS (+CI) ^(m)/z208 [M+H)⁺.

EXAMPLE 1(±)-(R*R*)-4′-Amino-5′-fluoro-1,3,4,10a-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)quinazoline]-6-one

A solution of 2-amino-6-fluorobenzamidine (0.13 g, 0.61 mmol) and1,3,4,10a-tetrahydro[pyrido[2,1-a]]isoindole-2,6-dione (0.12 g, 0.59mmol) (J.C.S. Perkin Trans. 1, 1984, 2477; Tetrahedron, 1993, 2239) inethanol (15 ml) was stirred at reflux for 18 h. The mixture was cooledand filtered to give a yellow solid (0.1 g), which was purified by flashcolumn chromatography on neutral alumina, eluting withdichloromethane/methanol (99:1 to 95:5) as eluent to afford a singlediastereoisomer of the title compound (containing the RR and SSenantiomers), as a yellow solid (0.05 g). M.p. 207-209° C.

EXAMPLE 2(±)-(R*S*)-4′-Amino-5′-fluoro-1,3,4,10a-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)quinazoline]-6-one

The filtrate obtained after the precipitation of the product of Example1 was concentrated to give a brown oil (0.19 g). Purification by flashcolumn chromatography on neutral alumina, eluting withdichloromethane/methanol (99:1 to 95:5) as eluent gave a seconddiastereoisomer of the title compound (containing the RS and SRenantiomers), as a yellow solid (0.03 g). M.p.184-186° C.

EXAMPLE 3(±)-(R*R*)-4-Amino-5-fluoro-3b′,4′,6′,7′-tetrahydrospiro[quinazoline-2,5′(2H,9′H-thieno[3,2-a]indolizine]-9′-one

A solution of 2-amino-6-fluorobenzamidine dihydrochloride (100 mg., 44mmol) and 3b,4,6,7-tetrahydrothieno[3,2-a]indolizine-5,9-dionePreparation 7, 80 mg, 39 mmol) in ethanol (10 ml) was heated at refluxfor 5 h The solvent was evaporated and the residue separated bychromatography on silica using dichloromethane-methanol mixtures aseluent. The first eluted fraction provided the title compound (63 mg,43%) as a pale yellow solid, m.p. 193-195° C. MS (+CI) ^(m)/z 343[M+H]⁺.

EXAMPLE 4(±)-(R*S*)-4-Amino-5-fluoro-3b′,4′,6′,7′-tetrahydrospiro[quinazoline-2,5′(2H,9′H)-thieno[3,2-a]indolizine]-9′-one

The second eluted fraction from the chromatography described in Example3 afforded the title compound (33 mg, 22%), m.p. 218-220° C. MS (+CI)^(m)/z 343 [M+H]⁺.

The compounds of Examples 5 to 30 were made by the method of Examples 3and 4 using the intermediates stated.

The compounds of Examples 5 and 6 were obtained from2-amino-6-fluorobenzamidine dihydrochloride and6,7,9,9a-tetrahydrothieno[2,3-a]indolizine-4,8-ione (Preparation 16).

EXAMPLE 5(±)-(R*R*)-4-Amino-5-fluoro-6′,7′,9′,9′a-tetrahydrospiro[quinazoline-2,8′(2H,4′H)-thieno[2,3-a]indolizine]-4′-one

Obtained as the hydrochloride salt, m.p. >275° C. MS (+CI) ^(m)/z 343[M+H]⁺.

EXAMPLE 6(±)-(R*S*)-4-Amino-5-fluoro-6′,7′,9′,9′a-tetrahydrospiro[quinazoline-2,8′(2H,4′H)-thieno[2,3-a]indolizine]-4′-one

Obtained as the hydrochloride salt, m.p. 225-230° C. MS (+CI) ^(m)/z 343[M+H]⁺.

The compounds of Examples 7 and 8 were obtained from2-amino-6-fluorobenzamidine dihydrochloride and7-chloro-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione(Preparation 4).

EXAMPLE 7(±)-(R*R*)-4′-Amino-7-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-oneHydrochloride

MS (+CI) ^(m)/z 371/373 M+H]⁺; 300 MHz ¹H NMR (d₆-DMSO) 7.62 (1H, d, J7.5 Hz), 7.54 (1H, t, J 7.5 Hz), 7.45 (1H, d, J 7.2 Hz), 7.13 (1H, dt, J6.6, 6.6, 8.1 Hz), 6.59 (1H, s), 6.40 (1H, d, J 7.2 Hz), 6.37-6.33 (1H,m), 5.85-5.8 (2H, br.s), 4.79 (1H, dd, J3.3, 11.4 Hz), 4.12 (1H, dd, J3.9, 13.8 Hz), 3.40-3.35 (1H, m), 2.60-2.55 (1H, m), 1.91 (1H, d, J12Hz), 1.37 (1H, dt, J 5.7, 12.9, 12.9 Hz), 1.04 (1H, t, J 11.7 Hz).

EXAMPLE 8(±)-(R*S*)-4′-Amino-7-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′H)-quinazoline]-6-onehydrochloride

MS (+CI) ^(m)/z 371/373 [M+H]⁺; 300 MHz ¹H NMR (d₆-DMSO) 7.61-7.46 (4H,m), 7.27-7.20 (2H, m), 6.74 (1H, d, J 8.1 Hz), 6.44 (1H, dd, J 8.4, 12.3Hz), 4.75 (1H, dd, J 3.6, 3.9 Hz), 3.32-3.19 (1H, m), 2.0-1.95 (1H, m),1.5-1.4 (1H, m), 1.3-1.1 (1H, m).

The compounds of Examples 9 and 10 were obtained from2-amino-6-fluorobenzamidine dihydrochloride and8-chloro-1,3,4,10b-tetrahydropyrido[(2,1-a]isoindole-2,6-dione(Preparation 6).

EXAMPLE 9 (±)(R*R*)-4-Amino-8-chloro-5′-fluoro-1,3,4,10b-tethydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt, m.p. 308° C. (dec.).

EXAMPLE 10(±)-(R*S*)-4′-Amino-8-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the trifluoroacetate salt (amorphous) after purification byreverse-phase HPLC. MS (+CI) ^(m)/z 371 [M+H]⁺; MS (−CI) ^(m)/z 369[M−H]⁺.

The compounds of Examples 11 and 12 were obtained from2-amino-6fluorobenzamidine dihydrochloride and9-chloro-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dionePreparation 15).

EXAMPLE 11(±)-(R*R*)-4′-Amino-9-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt, map. 225° C. (dec).

EXAMPLE 12(±)-(R*S*)-4′-Amino-9-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt, m.p. 210° C. (dec).

The compounds of Examples 13 and 14 were obtained from2-amino-6-fluorobenzamidine dihydrochloride and10-chloro-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione(Preparation 14).

EXAMPLE 13(±)-(R*R*)-4′-Amino-10-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinoline]-6-one

Obtained as the hydrochloride salt. MS (+CD) ^(m)/z 371 [M+H]⁺.

EXAMPLE 14(±)-(R*S*)-4′-Amino-10-chloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt MS (+CI) ^(m)/z 371 [M+H]⁺.

The compounds of Examples 15 and 16 were prepared from2-amino-6-fluorobenzamidine dihydrochloride and7,8-dichloro-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione(Preparation 5).

EXAMPLE 15(±)-(R*R*)-4′-Amino-7,8-dichloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt, m.p. 258-260° C.

EXAMPLE 16(±)-(R*S*)-4′-Amino-7,8-dichloro-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt, m.p. 259-261° C.

The compounds of Examples 17 and 18 were obtained from2-amino-6-fluorobenzarmidine dihydrochloride and7-methyl-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione(Preparation 11).

EXAMPLE 17(±)-(R*R*)-4′-Amino-7-methyl-5′-fluoro-1,3,4,10b-tetrhydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt, m.p. 201-204° C.

EXAMPLE 18(±)-(R*S*)-4′-Amino-7-methyl-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt, m.p. 215-218° C.

The compounds of Examples 19 and 20 were obtained from2-amino-6-fluorobenzamidine dihydrochloride and8-methyl-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione(Preparation 8).

EXAMPLE 19(±)-(R*R)-4′-Amino-8-methyl-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt, m.p. 238-239° C.

EXAMPLE 20(±)-(R*S*)-4′-Amino-8-methyl-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt, mp. 222-224° C.

The compounds of Examples 21 and 22 were prepared from2-amino-6-fluorobenzamidine dihydrochloride and8-methoxy-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione(Preparation 13).

EXAMPLE 21(±)-(R*R*)-4′-Amino-8-methoxy-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt MS (+CI) ^(m)/z 367 [M+H]⁺.

EXAMPLE 22(±)-(R*S*)-4′-Amino-8-methoxy-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt. MS (+CI) ^(m)/z ₃₆₇ [M+H]⁺.

The compounds of Examples 23 and 24 were obtained from2-amino-6-fluorobenzamidine dihydrochloride and8-cyano-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione(Preparation 9).

EXAMPLE 23(±)-R*R*)-4-Amino-8-cyano-5′-fluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt, m.p. 241° C. (dec).

EXAMPLE 24(±)-R*S*)-4′-Amino-8-cyano-5′-fluoro-1,3,4,10b-tetahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt, m.p. 290° C. (dec).

The compounds of Examples 25 and 26 were obtained from from²-amino-3,6-difluoro benzoamidine hydrochloride and8-cyano-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-dione(Preparation 9).

EXAMPLE 25(±)-(R*R*)-4′-Amino-8-cyano-5′,8′-difluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinazoline]-6-one

Obtained as the hydrochloride salt, m.p. 243° C. (dec).

EXAMPLE 26(±)-(R*S*)-4′-Amino-8-cyano-5′,8′-difluoro-1,3,4,10b-tetrahydrospiro[pyrido[2,1-a]isoindole-2,2′(1′H)-quinoline]-6-one

M.p. 180° C. (dec).

The compounds of Examples 27 and 28 were obtained from2-amino-6-fluorobenzamidine dihydrochloride and7,8,10,10a-tetahydropyrido[3,4-a]indolizine-5,9-dione (Preparation 10).

EXAMPLE 27(±)-(R*R*)-4-Amino-5′-fluoro-7,8,10,10a-tetrahydrospiro[pyrido[3,4-a]indolizine-9,2′(1′H)-quinazoline]-5-one

Obtained as the hydrochloride salt. MS (+CI) ^(m)/z 338 [M+H]⁺; 300 MHz¹H NMR (d₆-DMSO) 8.97 (1H, s), 8.72 (1H, d, J 5.1 Hz), 7.67 (1H, d, J 5Hz), 7.20 (1H, q, J 7 Hz), 6.8 (1H, br.s), 6.46-6.38 (2H, m), 4.97 (1H,dd, J 3.6, 12 Hz), 4.18 (1H, dd, 3 4.5 12.6 Hz), 3.45 (1H, dt, 3.3, 12.9Hz), 2.76-2.72 (1H, m), 1.97 (1H, d, J 12 Hz), 1.45 (1H, dt, J 5.4, 12.9Hz), 1.10 (1H, t, J 12.0 Hz).

EXAMPLE 28(±)-(R*S*)-4′-Amino-5′-fluoro-7,8,10,10a-tetrahydrospiro[pyrido[3,4-a]indolizine-9,2′(1′H)-quinazoline]-5-one

MS (+CI) ^(m)/z 338 [M+H]⁺; 300 MHz ¹H NMR (d₆-DMSO) 8.95 (1H, s), 8.74(1H, d, J 4.8 Hz), 7.69 (1H, d, J 5.1 Hz), 7.44-7.25 (3H, m), 6.78 (1H,d, J 7.5 Hz), 6.52-6.46 (1H, m), 4.94 (1H, dd, J 3.6, 12.3 Hz), 4.20(1H, dd, J 4.8, 13.8 Hz), 3.8-3.43 (1H, dd, obscured by H₂O), 2.71 (1H,br.d, J 9 Hz), 1.98 (1H, d, J 13.2 Hz), 1.55 (1H, dt, J 5.1, 12.9 Hz),1.26 (1H, t, J 12.9 Hz).

The compounds of Examples 29 and 30 were obtained from2-amino-6-fluorobenzamidine dihydrochloride and6b,7,9,10-tetrahydronaphtho[2,1-a]indolizine-8,12-dione (Preparation12).

EXAMPLE 29(±)-(R*R*)-4-Amino-5′-fluoro-6b,7,9,10-tetahydrospiro[naphtho[2,1-a]indolizine-8,2′(1′H)-quinazoline]-12-one

Obtained as the hydrochloride salt, m.p. 208-210° C.

EXAMPLE 30(±)-(R*S*)-4′-Amino-5′-fluoro-6b,7,9,10-tetrahydrospiro[naphtho[2,1-a]indolizine-8,2′(1′H)-quinazoline]-12-one

Obtained as the hydrochloride salt, m.p. 235-237° C.

Screens

The pharmacological activity of compounds according to the invention wastested in the following screens.

Screen 1

The activity of compounds of formula (I), or a pharmaceuticallyacceptable salt, enantiomer or tautomer thereof, may be screened fornitric oxide synthetase inhibiting activity by a procedure based on thatof Förstennann et al., Eur., Pham, 1992, 225, 161-165. Nitric oxidesynthase converts ³H-L-arginine into ³H-L-citrulline which can beseparated by cation exchange chromatography and quantified by liquidscintillation counting.

Enzyme is prepared, after induction, from the cultured murine macrophagecell line J774A-1 (obtained from the laboratories of the Imperial CancerResearch Fund). J774A-1 cells are cultured in Dulbeccos Modified EaglesMedium (DMEM) supplemented with 10% foetal bovine serum, 4 mML-glutamine and antibiotics (100 units/ml penicillin G, 100 mg/mlstreptomycin & 0.25 mg/ml amphotericin B). Cells are routinely grown in225 cm³ flasks containing 35 ml medium kept at 37° C. and in ahumidified atmosphere containing 5% CO₂.

Nitric oxide synthase is produced by cells in response to interferon-g(IFNg) and lipopolysaccharide (LPS). The medium from confluent cultureflasks is removed and replaced with 25 ml (per flask) of fresh mediumcontaining 1 mg/ml LPS and 10 units/ml IFNg. After a period of 17-20hours in culture, harvesting of cells is accomplished by scraping thecell sheet from the flask surface into the culture medium. Cells arecollected by centrifugation (1000 g for 10 minutes) and lysate preparedby adding to the cell pellet a solution containing 50 mM Tris-HCl (pH7.5 at 20° C.), 10% (v/v) glycerol, 0.1% (v/v) Triton-X-100, 0.1 mMdithiothreitol and a cocktail of protease inhibitors comprisingleupeptin (2 mg/ml), soya bean trypsin inhibitor (10 mg/ml), aprotinin(5 mg/ml) and phenylmethylsulphonyl fluoride (50 mg/ml).

For the assay, 25 μl of substrate cocktail (50 mM Tris-HCl (pH 7.5 at20° C.), 400 μM NADPH, 20 μM flavin adenine dinucleotide, 20 μM Ravinmononucleotide, 4 μM tetrahydrobiopterin, 12 μM L-arginine and 0.025 mCiL-[³H]arginine) is added to wells of a 96 well filter plate (0.45 μMpore size) containing 25 μl of a solution of test compound in 50 mMTris-HCl. The reaction is started by adding 50 μl of cell lysate(prepared as above) and after incubation for 1 hour at room temperatureis terminated by addition of 50 μl of an aqueous solution of 3 mMnitroarginine and 21 mM EDTA.

Labelled L-citrulline is separated from labelled L-arginine using DowexAG-50W. 150 μl of a 25% aqueous slurry of Dowex 50W (Na⁺ form) is addedto the assay after which the whole is filtered into 96 well plates. 75μl of filtrate is sampled and added to wells of 96 well platescontaining solid scintillant. After allowing the samples to dry theL-citrulline is quantified by scintillation counting.

In a typical experiment basal activity is 300 dpm per 75 μl sample whichis increased to 1900 dpm in the reagent controls. Compound activity isexpressed as IC₅₀ (the concentration of drug substance which gives 50%enzyme inhibition in the assay) and aminoguanidine, which gives an IC₅₀(50% inhibitory concentration) of 10 μM, is tested as a standard toverify the procedure. Compounds are tested at a range of concentrationsand from the inhibitions obtained IC₅₀ values are calculated. Compoundsthat inhibit the enzyme by at least 25% at 100 μM are classed as beingactive and are subjected to at least one retest.

In the above screen, the compounds of Examples 1 to 30 were tested andgave IC₅₀ values of less than 25 μM indicating that they are expected toshow useful therapeutic activity.

Screen 2

Compounds also show activity against the human form of induced nitricoxide synthase as can be demonstrated in the following assay.

Enzyme is prepared, after induction, from the cultured human colonadrenocarcinoma cell line DLD1 (obtained from the European Collection ofAnimal Cell Culture—cell line number 90102540). DLD1 cells are culturedin RPMI 1640 medium supplemented with 10% foetal bovine serum, 4 mML-glutamine and antibiotics (100 units/ml penicillin G, 100 μg/mlstreptomycin and 0.25 μg/ml amphotericin B). Cells are routinely grownin 225 cm³ flasks containing 35 ml medium kept at 37° C. and in ahumidified atmosphere containing 5% CO₂.

Nitric oxide synthase is produced by cells in response to interferon-γ(IFN-γ) and interleukin-1β (IL-1β). The medium from confluent flasks isremoved and replaced with 25 ml (per flask) of fresh medium containing250 units/ml IL-1β and 1000 units/ml IFN-γ. After a period of 17-20hours in culture, harvesting of cells is accomplished by scraping thecell monolayer from the flask surface into the culture medium. Cells arecollected by centrifugation (1000 g for 10 minutes) and lysate preparedby adding to the cell pellet a solution containing 50 mM Tris-HCl (pH7.5 at 20° C.), 10% (v/v) glycerol, 0.1% (v/v) Triton-X100, 0.1 mMdithiothreitol and a cocktail of protease inhibitors including leupeptin(2 μg/ml), soya bean trypsin inhibitor (10 μg/nil), aprotonin (5 μg/ml)and phenylmethylsulphonyl fluoride (50 μg/ml).

For the assay, 25 μl of substrate cocktail (50 mM Tri-HCl (pH 7.5), 400μM NADPH, 20 μM flavin adenine dinucleotide, 20 μM flavin mononucleotideand 4 μM tetrahydrobiopterin) is added to the wells of a 96-well plate.Test compounds are preincubated with enzyme by adding together with 40μl of cell lysate (prepared as above) and incubating for 1 hour at 37°C. at the end of which period 10 μl of 30 μM L-arginine and 0.025 μCi ofL-[³H]-arginine in 50 mM Tris-HCl is added to start the enzymaticreaction. Incubation is continued for a further 1 hour at 37° C. Thereaction is terminated by addition of 50 μl of an aqueous solution of 3mM nitroarginine and 21 mM EDTA.

Labelled L-citrulline is separated from labelled L-arginine using DowexAG-50W. 120 μl of a 25% aqueous slurry of Dowex 50W is added to 96 wellfilter plates (0.45 μm pore size). To this is added 120 μl of terminatedassay mix. 75 μl of filtrate is sampled and added to the wells of 96well plates containing solid scintillant. After allowing the samples todry the L-citrulline is quantified by scintillation counting.

In a typical experiment basal activity is 300 dpm per 75 μl sample ofreagent controls, which is increased to 3000 dpm in the presence ofenzyme. Compound activity is expressed as IC₅₀ (the concentration ofdrug substance which gives 50% enzyme inhibition in the assay) andL-NMMA, which gives an IC₅₀ of about 0.4 μM is tested as a standard toverify the procedure. Compounds are tested at a range of concentrationsand from the inhibitions obtained IC₅₀ values are calculated.

In this screen the compounds of Examples 1 to 30 give IC₅₀ values lessthan 25 μM, indicating that they are predicted to show usefulltherapeutic activity.

What is claimed is:
 1. A compound of formula (I)

wherein R¹ and R² independently represent hydrogen, C1 to 6 alkyl, C2 to6 alkenyl, C2 to 6 alkynyl, C1 to 6 alkoxy, C1 to 6 alkylthio, halogen,hydroxy, trifluoromethyl or amino; R³ represents one or moresubstituents independently selected from hydrogen, C1 to 6 alkyl, C2 to6 alkenyl, C2 to 6 alkynyl, C1 to 6 alkoxy, C1 to 6 alkylthio, halogen,hydroxy, trifluoromethyl, amino, cyano, nitro, trifluoromethoxy,methanesulphonyl, sulphamoyl, —NR⁴R⁵, —COOR⁶, —CONR⁷R⁸, benzyloxy,phenyl, or a 5-membered heterocyclic aromatic ring containing 1 to 3heteroatoms which may be the same or different and are selected from O,N and S, which phenyl or 5-membered heterocyclic aromatic ring isoptionally substituted, the optional substituents being C1 to 6 alkyl,halogen, cyano, nitro, hydroxy, C1 to 6 alkoxy, trifluoromethyl andtrifluoromethoxy; R⁴, R⁵ and R⁶ independently represent hydrogen or C1to 6 alkyl; R⁷ and R⁸ independently represent hydrogen, C1 to 6 alkyl orphenyl, which phenyl is optionally substituted by one or more groupsindependently selected from C1 to 6 alkyl, halogen, cyano, nitro,hydroxy, C1 to 6 alkoxy, trifluoromethyl and trifluoromethoxy; Xrepresents —(CH₂)_(n)—, wherein n represents zero or 1; and A representsa 5- or 6membered heterocyclic aromatic ring containing 1 to 3heteroatoms which may be the same or different and are selected from O,N and S; or a pharmaceutically acceptable salt, enantiomer, racemate ortautomer thereof.
 2. A compound of formula (I), according to claim 1,wherein R¹ represents hydrogen.
 3. A compound of formula (I), accordingto claim 1, wherein R¹ represents fluoro.
 4. A compound of formula (I),according to claim 1, wherein R² represents fluoro.
 5. A compound offormula (I), according to claim 1, wherein X represents —(CH₂)_(n)— andn represents zero.
 6. A compound of formula (I) which is:(R*R*)-4-amino-5-fluoro-3b′,4′,6′,7′-tetrahydrospiro[quinazoline-2,5′(2H,9′H)-thieno[3,2-a]indolizine]-9′-one;(R*S*)-4-amino-5-fluoro-3b′,4′,6′,7′-tetrahydrospiro[quinazoline-2,5′(2H,9′H)-thieno[3,2-a]indolizine]-9′-one;(R*R*)-4-amino-5-fluoro-6′,7′,9′,9a′-tetrahydrospiro[quinazoline-2,8′(2H,4′H)-thieno[2,3-a]indolizine]-4′-one;(R*S*)-4-amino-5-fluoro-6′,7′,9′,9a′-tetraydrospiro[quinazoline-2,8′(2H,4′H)-thieno[2,3-a]indolizine]-4′-one;(R*R*)-4-amino-5′-fluoro-7,8,10,10a-tetrahydrospiro[pyrido[3,4-a]indolizine-9,2′(1′H)-quinazoline]-5-one;(R*S*)-4′-amino-5′-fluoro-7,8,10,10a-tetrahydrospiro[pyrido[3,4-a]indolizine-9,2′(1′H)quinazoline]-5-one;or a pharmaceutically acceptable salt, enantiomer or tautomer thereof.7. A pharmaceutical composition comprising a compound of formula (I)according to any claim 1, or a pharmaceutically acceptable salt,enantiomer or tautomer thereof, in admixture with a pharmaceuticallyacceptable adjuvant, diluent or carrier.
 8. A method of treating, orreducing the risk of, human diseases or conditions in which inhibitionof nitric oxide syntase activity is beneficial which comprisesadministering a therapeutically effective amount of a compound offormula (I), as defined in claim 1, or a pharmaceutically acceptablesalt, enantiomer or tautomer thereof, to a person suffering from, or atincreased risk of, such diseases or conditions.
 9. A method of treatmentaccording to claim 8 in which it is predominantly inducible nitric oxidesynthase that is inhibited.
 10. A method of treating, or reducing therisk of, inflammatory disease in a person suffering from, or at risk of,said disease, wherein the method comprises administering to the person atherapeutically effective amount of a compound of formula (I), asdefined in claim 1, or a pharmaceutically acceptable salt, enantiomer ortautomer thereof.
 11. The method of treatment as claimed in claim 10wherein the disease is asthma or rheumatoid arthritis.
 12. A method oftreating, or reducing the risk of, pain in a person suffering from, orat risk of, said condition, wherein the method comprises administeringto the person a therapeutically effective amount of a compound offormula (I), as defined in claim 1, or a pharmaceutically acceptablesalt, enantiomer or tautomer thereof.
 13. A method of treating, orreducing the risk of, inflammatory disease in a person suffering from,or at risk of, said disease, wherein the method comprises administeringto the person a therapeutically effective amount of a combination of acompound of formula (I), as defined in claim 1, or a pharmaceuticallyacceptable salt, enantiomer or tautomer thereof, with a COX-2 inhibitor.14. A process for the preparation of a compound of formula (I), asdefined in claim 1, or a pharmaceutically acceptable salt, enantiomer ortautomer thereof, wherein the process comprises reaction of a compoundof formula (II), or a salt thereof:

wherein R¹ and R² are as defined in claim 1, with a compound of formula(III) or a protected derivative thereof:

wherein R³, A and X are as defined in claim 1.